Insights into the function of the chloroplastic ribosome‐associated GTPase high frequency of lysogenization X in Arabidopsis thaliana

Abstract Ribosome‐associated GTPases are conserved enzymes that participate in ribosome biogenesis and ribosome function. In bacteria, recent studies have identified HflX as a ribosome‐associated GTPase that is involved in both ribosome biogenesis and recycling under stress conditions. Plants possess a chloroplastic HflX homolog, but its function remains unknown. Here, we characterized the role of HflX in the plant Arabidopsis thaliana . Our findings show that HflX does not affect normal plant growth, nor does it play an essential role in acclimation to several different stresses, including heat, manganese, cold, and salt stress under the conditions tested. However, we found that HflX is required for plant resistance to chloroplast translational stress mediated by the antibiotic lincomycin. Our results suggest that HflX is a chloroplast ribosome‐associated protein that may play a role in the surveillance of translation. These findings provide new insight into the function of HflX as a ribosome‐associated GTPase in plants and highlight the importance of investigating conserved proteins in different organisms to gain a comprehensive understanding of their biological roles.


Introduction
How many HflX genes are in Arabidopsis?Does the number of HflX and HflX-like genes vary between different plant species?Results Page 8.During the analysis of the structural characteristics of the Arabidopsis HflX and HflX-like proteins, did authors look for possible phosphorylation sites given the observation that E. coli HflX apparently possess an autophosphorylation activity (Gosh et al., doi: 10.1002/2211-5463.12065FEBS 2016) Page 9.After evaluating a rosette growth through the measurement of its size in wild type and mutant lines, authors concluded that HflX does not influence Arabidopsis normal growth and development.However, testing just this phenotype is not enough to reach such wide conclusion.Authors do not mention if they evaluated germination rate, leaf size, flowering time, seed size, yield, root architecture, etc, etc. Authors should assess additional phenotypes during different developmental stages to determine whether there is or not any effect on growth and development in Arabidopsis plants lacking HflX.Maybe checking those traits where an active translation is needed would a good start.
Page 11.Do authors compare basal heat tolerance of the mutants, or other phenotypes to look at the mutant performance through different heat plant responses?Do authors consider that the evaluation they did is enough to be certain that HflX does not participate in acclimation to HS? Page 11.Regarding manganese effects, just one manganese concentration (2 mM) was tested for this evaluation?Why was 2 mM manganese used in these experiments?A manganese concentration sensitivity curve should be done including all lines.Why did authors choose this plant developmental stage for the different evaluations?Page 13.Even though, the authors did not find differences in the 23S/18S ratio between wild type and hflX mutant lines, still it is possible that the rRNAs were not correctly assembled, and therefore translation was not efficient enough.Chloroplast translation efficiency should be assessed to look for an effect on ribosome reconstitution or functionality.

Reviewer #3:
GTPases assist ribosome assembly, translation, and turnover in prokaryotes and animal cells.Chloroplast have proteins homologous to bacterial HflX proteins have been identified and shown to bind the 50S ribosomal subunit, although their role in ribosome function is unknown.This study focuses on the physiological function of plastid HflX through the generation of T-DNA insertion lines lacking HflX expression and function.HflX was identified as a nuclear encoded gene as well as identifying a second nuclear gene HflX-like.The current study focuses on HflX.The authors demonstrate that interruption of hflx does not seem to be required for normal growth.In addition, through a battery of stresses that are known to affect chloroplast translation (heat shock, cold treatment, high Mn2+, high salt), hflx plants grew comparable to wildtype plants.However, when treated with lincomycin, a drug that interferes with prokaryotic translation by blocking the peptidyl transferase center, the hflx plants were more severely affected than wildtype, suggesting a possible role for HflX in ribosome splitting or recycling.It would have strengthened the manuscript to also include studies on Hflx-like protein as well, but I look forward to seeing characterization of that protein in future work.
Overall, this manuscript is well-written and presents sound experiments in a logical way.I have some minor issues to be addressed by the authors.
• In the cartoon domain models of cpHflX and E. coli HflX, what does the red region signify?Why is it designated separately from the rest of the NTD? • Line 227 and Figure S1: please indicate the enlarged N-loop of the NTD of HflX-like protein.It is difficult to distinguish the enlarged N-loop from the N-loops of HflX protein.The images look more like they are at different scales, which would also give the impression that the N-loop of HflX-like is enlarged.
• Figure 2: Can the authors offer an explanation as to why the DNA in hflx1-3 appears to be less than the other lines and wildtype?• Figure 4C: What about hflx1-3?Did hflx1-3 also show no significant difference between the 23S/18S rRNA ratios?Your article will appear online in the next available issue of Plant Direct.To ensure your article gets published as quickly as possible, please pay attention to the steps detailed below.We have found that most of the delays happen at this stage, especially at the payment stage, so please respond as quickly as possible when prompted.
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In our phylogenetic analysis, we selected plants and algae representative of certain groups (monocots, dicots etc) and show all the HflX or HflX-like that are present in the genomes of these plants.All plants analyzed have a single copy of HFLX and HFLX-like, except for P. patens where HFLX-like is absent.Although the phylogenetic analysis would suggest that there is usually one of each, we cannot exclude that other plants not tested have different HFLX or HFLX-like copy numbers.We now mention the observed HFLX and HFLX-like copy number in the introduction and in the results.Results 1.2.Page 8.During the analysis of the structural characteristics of the Arabidopsis HflX and HflX-like proteins, did authors look for possible phosphorylation sites given the observation that E. coli HflX apparently possess an autophosphorylation activity (Gosh et al., doi: 10.1002/2211-5463.12065FEBS 2016) > Ser211, the site of autophosphorylation of E. coli HflX, is conserved in plant HflX and HflX-like.In C. reinhardtii HflX-like, the Ser is substituted with a Thr.We now mention this in the results.1.3.Page 9.After evaluating a rosette growth through the measurement of its size in wild type and mutant lines, authors concluded that HflX does not influence Arabidopsis normal growth and development.However, testing just this phenotype is not enough to reach such wide conclusion.Authors do not mention if they evaluated germination rate, leaf size, flowering time, seed size, yield, root architecture, etc, etc. Authors should assess additional phenotypes during different developmental stages to determine whether there is or not any effect on growth and development in Arabidopsis plants lacking HflX.Maybe checking those traits where an active translation is needed would a good start.
> We agree that our wording is too wide given that we did not quantify a wider range of plant growth parameters.We note that we didn't observe visible differences in germination rate, leaf size (pictured) or flowering time.However, we did not quantify these observations.We also note the rosette growth is usually slower in the presence of chloroplast translation defects (e.g.Meteignier et al. 2021 10.1093/nar/gkaa1244).We have modified the text in the abstract, results and discussion sections to better constrain our claims.
1.4.Page 11.Do authors compare basal heat tolerance of the mutants, or other phenotypes to look at the mutant performance through different heat plant responses?Do authors consider that the evaluation they did is enough to be certain that HflX does not participate in acclimation to HS? > EcHflX was previously shown to be essential in response to heat-shock in E. coli (Zhang et al. 2015 https://doi.org/10.1038/nsmb.3103).This led us to test a comparable experimental setting in Arabidopsis where we do not observe an essential requirement for HflX.We did try a range of different temperatures and time exposures (not shown) but we always obtained a similar phenotype to the WT.
We are not sure that any scientific result can demonstrate certainty, and this even more challenging in the case of negative results (e.g what about 41oC?Or 40.5? or 40.25? or 12h? etc).Regarding the contribution of HflX we have moved from maybe involved to probably not involved.We have modified the text to make this clearer.1.5.Page 11.Regarding manganese effects, just one manganese concentration (2 mM) was tested for this evaluation?Why was 2 mM manganese used in these experiments?A manganese concentration sensitivity curve should be done including all lines.Why did authors choose this plant developmental stage for the different evaluations?> We have found that if we want to find a sensitivity phenotype then it is good to treat plants with a condition that has a clear but not too severe consequence on growth (or other measured parameter).From our own preliminary experiments, and those of other researchers (Zhao et al. 2017 https://doi.org/10.3389/fpls.2017.00272) 2 mM manganese is the lowest concentration that gives a clear phenotype in the wild type.At higher concentrations we might miss potential changes in sensitivity due to the severity of the phenotype.
We used actively growing seedlings for our stress tests because this is when chloroplast biogenesis and translation are particularly active and it is also a convenient growth stage for controlled treatments and phenotype quantification.
1.6.Page 13.Even though, the authors did not find differences in the 23S/18S ratio between wild type and hflX mutant lines, still it is possible that the rRNAs were not correctly assembled, and therefore translation was not efficient enough.Chloroplast translation efficiency should be assessed to look for an effect on ribosome reconstitution or functionality.> We agree with the reviewer's comment.We mentioned this point in the discussion section (original line 358-363, revised line 368-373).
Reviewer #3: GTPases assist ribosome assembly, translation, and turnover in prokaryotes and animal cells.Chloroplast have proteins homologous to bacterial HflX proteins have been identified and shown to bind the 50S ribosomal subunit, although their role in ribosome function is unknown.This study focuses on the physiological function of plastid HflX through the generation of T-DNA insertion lines lacking HflX expression and function.HflX was identified as a nuclear encoded gene as well as identifying a second nuclear gene HflX-like.The current study focuses on HflX.The authors demonstrate that interruption of hflx does not seem to be required for normal growth.In addition, through a battery of stresses that are known to affect chloroplast translation (heat shock, cold treatment, high Mn2+, high salt), hflx plants grew comparable to wildtype plants.However, when treated with lincomycin, a drug that interferes with prokaryotic translation by blocking the peptidyl transferase center, the hflx plants were more severely affected than wildtype, suggesting a possible role for HflX in ribosome splitting or recycling.It would have strengthened the manuscript to also include studies on Hflx-like protein as well, but I look forward to seeing characterization of that protein in future work.
Overall, this manuscript is well-written and presents sound experiments in a logical way.I have some minor issues to be addressed by the authors.> We have modified Fig. S1 and Fig. 1 to illustrate the N-loop.We also checked the images and confirm that the scales are identical for the five structure representations in Fig. S1 (the images are produced from a single ChimeraX file containing the aligned 3D structures).
2.3.Figure 2: Can the authors offer an explanation as to why the DNA in hflx1-3 appears to be less than the other lines and wildtype?> This is a qualitative PCR assay and the difference is likely due to small differences in the starting DNA concentration.

DECISION LETTER-ROUND 2 :
December 7, 2023 Dr. Ben Field CNRS, CEA, University of Aix-Marseille BIAM 163 av de Luminy Marseille, N/A 13009 France MSID: 2023-01144R1 MS TITLE: Insights into the function of the chloroplastic ribosome-associated GTPase HflX in Arabidopsis thaliana.Dear Dr. Ben Field: I am pleased to inform you that your manuscript "Insights into the function of the chloroplastic ribosome-associated GTPase HflX in Arabidopsis thaliana."has been accepted for publication in Plant Direct.
-Reviewer comments: Introduction 1.1.How many HflX genes are in Arabidopsis?Does the number of HflX and HflX-like genes vary between different plant species?> In the introduction we mention the known HflX reported in plants (Suwastika et al., 2014).

2. 1 .
In the cartoon domain models of cpHflX and E. coli HflX, what does the red region signify?Why is it designated separately from the rest of the NTD? > We apologize for not explaining the colors in the legend.We used the E. coli HflX model presented previously (Zhang et al. 2015 https://doi.org/10.1038/nsmb.3103).The NTD domain contains a nucleotide binding domain marked in blue and a helical domain marked in red.We have modified the legends of Fig. 1 and Fig. S1 accordingly.2.2.Line 227 and Figure S1: please indicate the enlarged N-loop of the NTD of HflX-like protein.It is difficult to distinguish the enlarged N-loop from the N-loops of HflX protein.The images look more like they are at different scales, which would also give the impression that the N-loop of HflX-like is enlarged.
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